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Development, Quantification, Method Validation and Stability Study of a Novel Fucoxanthin-Fortified Milk Il-Kyoon Mok, Jungro Yoon, Cheol-Ho Pan, and Sang Min Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02206 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016

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Journal of Agricultural and Food Chemistry

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Development, Quantification, Method Validation and Stability Study of

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a Novel Fucoxanthin-Fortified Milk

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Il-Kyoon Mok†,§, Jung-Ro Yoon§, Cheol-Ho Pan†, Sang-Min Kim†, ‡, *

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†

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Gangneung, Gangwon-do 25451, Republic of Korea

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‡

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Products, Gangneung, Gangwon-do 25451, Republic of Korea

Systems Biotechnology Research Center, KIST Gangneung Institute of Natural Products,

Convergence Research Center for Smart Farm Solution, KIST Gangneung Institute of Natural

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§

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Gangneung, Gangwon-do 210-702, Republic of Korea

Department of Food Processing and Distribution, Gangneung-Wonju National University,

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*Corresponding author:

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Sang-Min Kim, Tel:+82-33-650-3640, Fax: +82-33-650-3679

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E-mail:

[email protected]

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ACS Paragon Plus Environment


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■ABSTRACT

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In order to extend the scope of application of fucoxanthin, a marine carotenoid, whole milk (WM)

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and skimmed milk (SM) were fortified with fucoxanthin isolated from microalga Phaeodactylum

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tricornutum for a final 8 µg/mL milk solution concentration. Using these liquid systems, a

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fucoxanthin analysis method implementing extraction and HPLC-DAD was developed and validated

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by accuracy, precision, system suitability and robustness tests. The current method demonstrated

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good linearity over the range of 0.125 ~ 100 µg/mL of fucoxanthin with R2 = 1.0000 and all

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validation data supported the adequateness for the use in fucoxanthin analysis from milk solution. In

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order to investigate fucoxanthin stability during milk production and distribution, fucoxanthin

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content was examined during storage, pasteurization and drying processes under various conditions.

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Fucoxanthin in milk solutions showed better stabilizing effect in one month of storage period.

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Degradation rate constant (k) on fucoxanthin during this storage period suggested that fucoxanthin

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stability might be negatively correlated with decrease of temperature and increase of protein content

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such as casein and whey protein in milk matrix. In a comparison between SM and WM, fucoxantin

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in SM always showed better stability than that in WM during storage and three kinds of drying

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process. This effect was also deduced to relate with proteins content. In pasteurization step, more

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than 91% of fucoxanthin was retained after three pasteurization processes even though above trend

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was not found. This study demonstrated for the first time that milk products can be used as a basic

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food matrix for fucoxanthin application and protein content in the milk is an important factor for

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fucoxanthin stability.

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KEYWORDS: fucoxanthin; carotenoid analysis; stability; whole milk; skimmed milk

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■INTRODUCTION

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Fucoxanthin, one of the main marine carotenoids, contributes to more than 10% of the total

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carotenoids in nature.1 This carotenoid has been reported to exhibit various beneficial biological

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properties such as protective effects on the liver, as well as anti-oxidant, anticancer, anti-

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inflammatory, anti-diabetic, anti-angiogenic, anti-malarial, neuro-protective, and anti-obesity

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activities.2 Among them, the anti-obesity effect is the most prominent biological activity and a

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number of reports have supported this fact.3 Industrially, fucoxanthin has been produced from

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macroalgae such as Laminaria japonica and Undaria pinnatifida.4 Recently, microalga

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Phaeodactylum tricornutum has been suggested as another source for fucoxanthin production.5

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Although fucoxanthin can be produced industrially, its application in the food industry is limited

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for several reasons. On one hand, the unique molecular structure of fucoxanthin—including the

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unusual allenic bond, a 5,6-monoepoxide, and 9 conjugated double bonds—is susceptible to

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oxidation and isomerization. Thus, fucoxanthin would be unstable when exposed to heat, light,

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oxygen, metals, enzymes, unsaturated lipids and other pro-oxidant molecules.6, 7 On the other hand,

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the low pH and water-based environment in the digestive system often make it difficult for

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fucoxanthin to be absorbed in the small intestine.8 In order to overcome this drawback, several

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processing methods for fucoxanthin have been attempted using canola oil, biodegradable chitosan-

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glycolipid, and cetyl palmitate-canola oil mixed with a solid lipid core of fish gelatin..7, 9, 10 The

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stability and bioavailability of fucoxanthin was significantly improved by these functional

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encapsulation materials even though industrial process of production should be more discussed.

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Milk is an effective vehicle for micronutrients and is a good food system model for carotenoid 3



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application. Whey protein, β-lactoglobulin, and casein isolated from milk have been widely used as

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encapsulation materials for a number of bioactive substances including carotenoids.

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combination of sodium caseinate and whey protein isolate was used as a stabilizer for β-carotene in

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an oil-in-water emulsion system, demonstrating the highly increased stability of the pigment under

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these conditions.11 β-Lactoglobulin was also used as a vector for β-carotene, and its absorption was

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highly increased in in-vivo.12 Astaxanthin incorporation in different types of milk—whole milk

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(WM), semi-skimmed milk (SSM), and skimmed milk (SM)—showed that the retention of

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astaxanthin during storage decreased according to the increase of the fat content.13

11–13

A

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The fact that milk constituents bind strongly to carotenoid, as demonstrated by milk being a

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suitable vehicle for the pigment, can be a source of difficulty in carotenoid analysis. Some

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carotenoids such as lutein and zeaxanthin naturally occurring in milk and formulas are treated with a

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saponification or an enzymatic hydrolysis step because of the high fat content of the matrices.14

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However, these processes cause carotenoid losses of up to 40%. Thus, several modification methods

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have been developed for a more precise and accurate measurement of carotenoids in milk products.

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For instance, Yuhas et al.15 changed the order of extraction and saponification steps to recover more

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lipophilic carotenoids and used echinenone as an internal standard for the correction of losses.

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The aim of this study is to prepare value-added milk products by fucoxanthin fortification of WM

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and SM. Milk is generally considered the ideal food that contains the most nutrients for human

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health. Until now, a range of vitamins (vitamins A and D), minerals (iron, calcium, copper and zinc),

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and other nutrients such as polyunsaturated fatty acids have been used to fortify milk and provide

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various biological advantages.16 However, in fortification of milk by such function-inducing

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materials, several points should be confirmed prior to commercial production. The added nutrients

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should be stable during the storage period under the appropriate conditions and during industrial 4


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processing steps such as drying, pasteurization, and fermentation adopted to prepare dried milk

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powder or yogurt.

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Thus, in this study, we developed and validated fucoxanthin analysis methods for WM and SM

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based on a previously reported carotenoid analysis method.17 Additionally, the stability of

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fucoxanthin within the WM and SM matrices was investigated under the appropriate storage,

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pasteurization, and drying conditions.

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■ MATERIALS AND METHODS

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Materials. Fucoxanthin was purified from ethanol extract of microalgae Phaeodactylum tricornutum

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by silica-gel chromatography as described by Kim et al.5 and its purity was identified to be over 95%

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by NMR. Astaxanthin (purity ≥ 97%, HPLC grade) was purchased from Sigma-Aldrich. Two kinds

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of milk powders (WM, SM from Seoul Daily Cooperative, Korea) used as a model food matrix were

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purchased from the local market (Gangneung, Korea). The analytical grade extraction solvents

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including tert-butyl methyl ether (TBME) and petroleum ether were purchased from Sigma-Aldrich.

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Water and methanol with HPLC grade from Fisher Scientific Korea Ltd. were used for HPLC

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analysis.

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Fucoxanthin Fortification of Milk. In order to fortify fucoxanthin, fucoxanthin stock solution (4

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mg/mL in ethanol) was prepared with purified fucoxanthin. Milk solutions from WM and SM were

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prepared by mixing each powder with distilled water according to manufacturer’s suggestion (12.15%

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for WM powder, 12.27% for SM powder in water (w/w)). Fucoxanthin stock solutions were added to

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each milk solution while stirring the milk solution with a magnetic bar to make a final concentration 5



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of 8 µg/mL, corresponding to the recommended daily intake of fucoxanthin (2 mg in 250 mL milk

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solution). This concentration is also the minimum level of fucoxanthin demonstrated to have an anti-

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obesity effect in humans.18 Distilled pure water was used as a negative control for the matrix.

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Development of Fucoxanthin Extraction from Milk Solution. Fucoxanthin extraction from a milk

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solution was developed based on carotenoid analysis methods for foods.17 Briefly, the milk solution

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was deproteinated with ethanol, and then, fucoxanthin was extracted with petroleum ether and

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TBME solvents. In WM solution, an additional extraction step with hexane and 90% aqueous ethanol

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was added to remove milk fat. The steps are details as follows: 2 mL of fucoxanthin-fortified milk

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solution was mixed with the same volume of ethanol for deproteination and then, 1 mL of petroleum

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ether and TBME solvents was serially added to sample tube. The tube was vortexed for 30 seconds

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and centrifuged at 3,500 rpm for 5 min. to extract fucoxanthin from the food matrix. The supernatant

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was collected in an 8 mL glass vial. This extraction step was repeated three times and the collected

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extract solution was dried under N2 gas. The sample was dissolved in 1 mL of 90% aqueous ethanol

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and filtrated with 0.45 µm nylon membrane filter for HPLC analysis. In case of WM solution, milk

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fat was further removed from sample solution by extraction with 1 mL hexane solvent. The solution

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in ethanol phase was directly used in HPLC analysis following 0.45 µm nylon membrane filtration.

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HPLC Analysis. Quantification of fucoxanthin was performed by Agilent HPLC-DAD system

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(Agilent 1200 series, USA). YMC C-30 carotenoid column (250 × 4.6 mm ID, 3 µm particle size,

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Waters, Ireland) was used for the separation. Methanol and water solvent system was used for mobile

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phase at a flow rate of 0.7 mL/min with a column temperature of 35 ℃. The solvent gradient

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program was as follows: methanol/water ratio was increased from 90:10 to 100:0 over 20 min, and

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then, 100% methanol was held for the next 5 min. The chromatogram obtained at 450 nm was used

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for quantitative analysis of fucoxanthin (Fig. 1A). 6


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Validation of the Fucoxanthin Analysis Method. All validation tests except for accuracy test were

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performed with fucoxanthin stock solution dissolved in ethanol, but the accuracy test was assayed

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using fucoxanthin dispersed into milk solutions. The limit of detection (LOD) and the limit of

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quantification (LOQ) of fucoxanthin were measured with HPLC quantification at 10 concentration

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points from 0.125 to 100 µg/mL in ethanol. LOD and LOQ were carried out in triplicate. Precision

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data were obtained from the three stock solutions of the following fucoxanthin concentrations: 10, 20,

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and 50 µg/mL. Accuracy tests were carried out with two milk products fortified with fucoxanthin in

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two different concentrations (20 and 50 µg/mL) by calculating the fucoxanthin recovery value in

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terms of intra-day and inter-day variation. The precision and accuracy experiments were repeated 5

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times.19 The system suitability test was performed by analyzing the mixed stock solution of

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fucoxanthin and astaxanthin (as an internal standard) with each concentration of 20 µg/mL in ethanol.

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The developed HPLC-DAD method was used for 8 times repetition analysis and the RSD % of peak

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area and retention time, tailing factor, plate number and resolution were calculated with the analyzed

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HPLC chromatograms. The system suitability data was evaluated by the criteria of United States

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Pharmacopeia.20, 21 The robustness test was performed by changing the chromatographic conditions

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with ± 5% modifications each time in three HPLC analytical parameters (flow-rate (0.6 ~ 0.8

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mL/min), mobile phase % (methanol : water = 88 : 12 ~ 92 : 8) and oven temperature (33 ~ 37 ℃)).

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Three fucoxanthin stock concentrations (5, 10 and 20 µg/mL) were analyzed by HPLC and the result

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was evaluated by one-way ANOVA analysis of the fucoxanthin recovery percentage. 22, 23

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Stability Tests of Fucoxanthin in Milk Matrices. Fucoxanthin stability under storage conditions,

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pasteurization, and drying methods was investigated with fucoxanthin-fortified milk solutions. Three

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different temperatures (2, 10, and 26 ℃) were selected as the storage temperatures and milk 7



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solutions were stored in the dark. Samples were harvested weekly for fucoxanthin analysis for 2-4

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weeks. For pasteurization of milk solution, three different heat treatment methods were applied: Low

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Temperature Long Time (LTLT), 65 ℃/30 min; Hot Temperature Short Time (HTST), 75 ℃/15 sec;

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Ultra-High Temperature (UHT), 135 ℃/2 sec. Sample solutions were packaged into 2 mL glass vial

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and thermal treatment was carried out in an oil-bath. Thermal treatment time started when the

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solution reached a target temperature. For drying processes, two milk solutions were treated by hot-

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air drying, spray-drying, and freeze-drying methods. The equipment model and drying conditions

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were as follows: hot-air dryer (EYELA, WFO-400, temperature = 45 ℃, drying time = 48 hr), spray-

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dryer (OHKAWARA KAKOHKI, L-8, Inlet temperature = 180 ℃, Outlet temperature = 105 ℃,

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DISC rpm = 25,000), freeze-dryer (Operon, FDCF-12003, Coil temperature = –120 ℃, drying time =

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72 hr). After drying, each powder was re-dissolved to original ratio with water and fucoxanthin

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amount was quantified by HPLC.

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Kinetics Study of Fucoxanthin Degradation and Statistical Analysis. From the result of

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fucoxanthin storage in milk solutions (Fig. 2), fucoxanthin degradation curves were kinetically

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analyzed applying three equations of kinetic order (zero order: C = C0 – kt,

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kt, second order: 1/C = 1/C0 + kt), where C is fucoxanthin content (µg/mL) at storage time t; C0 is

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fucoxanthin content (µg/mL) at initial time; t is storage time (day); and k is degradation constant rate.

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Finally, the best equation was determined by the highest k-value and correlation coefficient (R2). The

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selected kinetic equation was used to evaluate the degradation rate (k) of fucoxanthin in milk

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solutions under the selected storage conditions.

first order: lnC = lnC0 –

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All experiments were performed in triplicate. All data are expressed as mean ± SD. Data was

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analyzed by t-test and one-way or two-way ANOVA (P < 0.05). The significant differences among 8


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means were separated by Duncan’s multiple range tests. All statistical analysis was processed using

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IBM SPSS 23.0 package and Microsoft office professional plus 2010 Excel.

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■ RESULTS AND DISCUSSION

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Optimization of Fucoxanthin Extraction Method from Milk Solution. There have been various

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carotenoid extraction methods from food products and most extraction methods generally follow

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three common steps: the release of carotenoid from food matrices by disrupting tissue, removal of

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unwanted components, and carotenoid concentration by liquid-liquid or liquid-solid extraction.17

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Milk also contains small amount of carotenoids such as lutein and zeaxanthin and several studies

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have developed analytical methods of carotenoids naturally found in milk matrices.14,15 In addition,

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the stability of astaxanthin is also used in milk products as a natural colorant was investigated under

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various conditions. As milk and related dairy products have more fat and protein content relative to

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common fruits and vegetables, carotenoid analysis methods based on the latter food models are

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different in terms of removing unwanted components. In this study, fucoxanthin was used to fortify

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two milk products (WM and SM)

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recommended daily intake amount of fucoxanthin (as 2 mg in 250 mL milk solution) and its

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analytical method was optimized for these food systems using previously reported methods.18, 24, 25

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Even though there have been several reports on fucoxanthin extraction from macro- or microalgaes,

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the extraction properties of fucoxanthin from the reported matrices are quite different from that of

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milk products.26, 27 As the first step, the volume ratio between ethanol and aqueous milk solution was

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optimized in a deproteination procedure. This step is necessary to separate the extraction phase

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(organic solvent) from the aqueous phase of the milk solution without gelation, which is an issue in

with final concentrations of 8 µg/mL corresponding to the

9



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the carotenoid analysis process using food systems containing high protein content such as milk

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products.17 Among the four tested volume ratios of ethanol (0.5, 1, 1.5 and 2) to the aqueous phase,

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the 1:1 ratio of ethanol and milk solution showed the best recovery yield for fucoxanthin (The

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fucoxanthin recovery % from milk solution (volume ratio of ethanol to milk solution) : 68.77% (0.5),

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95.51% (1), 80.13% (1.5), 56.53% (2)). As the next step, various organic solvents were investigated

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for the extraction efficiency of fucoxanthin from milk matrices. A wide variety of solvent

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combinations such as acetone/dichloromethane, acetone/ethanol, acetone/hexane, acetone/petroleum

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ether, and n-hexane/diethyl ether have been used as carotenoid extraction solvents for food

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systems.28-30 In this study, TBME, petroleum ether, dichloromethane, diethyl ether, n-hexane, and

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ethyl acetate were selected as the extraction solvents and their various combinations were tested in

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respect to the recovery yield of fucoxanthin from the milk solution. The mixed solvent of petroleum

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ether and TBME in equal parts was selected as the most appropriate extraction solvent system for

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fucoxanthin from milk products (data not shown).

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Among three model systems (WM, SM and water) fortified with fucoxanthin, SM and water

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showed high recovery yields of fucoxanthin (over 95% of the initial added amount) after three

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extraction steps. However, the recovery was significantly low in WM (45.29%) indicating that the

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extraction step with petroleum ether and TBME was not sufficient for the higher fat content solution.

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Following the drying process with N2 gas, the vial sample from WM exhibited a sticky property,

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which was not found in SM and water. Re-suspending the residue in the vial with 90% aqueous

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ethanol for HPLC analysis was not successful and led to a low recovery yield. As this phenomenon

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was attributed to the milk fat (milk fat content 27 g/100 g powder for WM, 1 g/100 g powder for

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SM), an additional solvent partition step with n-hexane was applied after the extraction step. Fig. 1B

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shows the effect of the n-hexane partition step in fucoxanthin extraction from the three systems. The

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recovery of fucoxanthin from WM was increased up to 89.83% from 45.29%. This result 10


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demonstrates that non-polar fat components were moved from the aqueous ethanol phase to the n-

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hexane phase. However, this additional step influenced negatively impacted the results for SM and

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water as a small amount of fucoxanthin moved from the aqueous ethanol phase to the n-hexane phase

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and recovery yields were decreased below 90%. Therefore, this additional solvent partition step was

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applied only to WM. Meanwhile, the saponification step did not influence the extraction yield in this

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study (data not shown). Though xanthophylls such as lutein and zeaxanthin require a saponification

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procedure for their release from esterified xanthophylls before extraction, fucoxanthin in algae does

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not naturally exist in the esterified form.31 For this reason, the saponification step was not included in

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the fucoxanthin analysis method of this study.

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Validation of Fucoxanthin Analysis Method from Milk Solution. The quantitative analysis

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method of fucoxanthin using HPLC was validated by various tests of linearity, limit of detection

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(LOD), limit of quantification (LOQ), precision, accuracy, system suitability and robustness test. The

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linearity of regression equation was determined by the analysis of fucoxanthin standard solution with

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concentrations in the range from 0.125 to 100 µg/mL. Table 1 shows the regression equations for

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fucoxanthin with a correlation coefficient of R2 = 1.0000, indicating good linearity. The LOD and

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LOQ values in HPLC analysis were calculated as the minimum concentration at the signal-to-noise

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(S/N) ratio equal to 3.3 and 10 and those of fucoxanthin were 0.027 and 0.082 µg/mL, respectively.

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Precision data obtained with three different concentrations of fucoxanthin standard solution (10, 20

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and 50 µg/mL) during a single day (intra-day) and over the course of 5 days (inter-day) shows

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relative standard deviation (RSD) values under 2.2%. In addition, the accuracy test by spiking WM

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and SM with two known concentrations of fucoxanthin (20 and 50 µg/mL) shows a minimum

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recovery value of 96.31% when the extracted fucoxanthin values in Fig. 1B were assumed to be 100%

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(Table 1). The system suitability result showed 0.24~0.98% RSD of peak area and retention time, 11



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plate number 27720, 1.0 of tailing factor, and 10.24 of resolution (Table S1). This means that the

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HPLC system and procedure is providing data with acceptable quality. The robustness data was

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obtained by slightly changing flow rate, mobile phase and column temperature coditions.

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Fucoxanthin recovery values (98.02~102.93%) at three concentration levels (5, 10, and 20 µg/mL)

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were not significantly affected by these changes of the HPLC condition (Table S2). These results

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indicate that the optimized extraction and HPLC analysis methods for fucoxanthin in milk solutions

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used in this study are valid and can be employed to access fucoxanthin content in milk products.

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Stability of Fucoxanthin in Milk Solutions and a Kinetic Study. Milk collected from a stock farm

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is usually stored in a big tank kept at 2 ℃ for a maximum of one month before food processing.

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Following food processing, milk products are commonly distributed and arranged on the grocery’s

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dairy case (open shelf) maintained at 10 ℃ for one week. In this study, fucoxanthin in milk solutions

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(WM and SM) was monitored at these two temperatures during storage periods to investigate its

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stability in the storage process. As a negative control, fucoxanthin stability was monitored at room

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temperature (26 ℃) for two weeks; this (storage at 26 ℃) was a relatively short period due to the

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decay of the milk. As seen in Fig. 2, fucoxanthin was quite stable in both milk solutions (WM and

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SM) in all tested temperature (below 20% of degradation), even though the degradation trend was

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slightly increased with increases in temperature. However, fucoxanthin in water exhibited a clear

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trend of instability with temperature increases. This result strongly suggests that fucoxanthin stability

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is enhanced by some components in the two milk solutions that are absent from water. At room

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temperature, almost 70% of added fucoxanthin in water was decomposed in two weeks (Fig. 2C).

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Fucoxanthin in SM showed slightly more stability than WM.

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In order to understand fucoxanthin degradation kinetics, the fucoxanthin samples from the 12


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storage stability test were analyzed through three equations of kinetic order. Among the zero to

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second order equations, the zero order equation showed the most accurate result in terms of the R2

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values and the degradation rate constant (k) as presented in Table 2. However, degradation of

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astaxanthin and ß-carotene has been demonstrated to follow first order kinetics in milk solutions and

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systems stabilized with milk proteins.6,13 The k values in Table 2 reveal two clear increasing

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tendencies based on the food matrix and the temperature. In addition, there is a significant gap in the

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k values between milk systems and water. These results follow the trends of the fucoxanthin

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degradation graphs in Fig. 2.

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The solution of astaxanthin in WM degraded more than that of SM because fat acids present in

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WM make astaxanthin more difficult to diffuse in the solution and form micelles with milk

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proteins.13, 32 Conversely, astaxanthin can interact more easily with milk proteins in SM, and can

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achieve stability more efficiently by forming micelles. Therefore, fat content is major factor for the

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stability difference between WM and SM. Another point to be considered is protein content in the

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milk solutions. As SM is produced by removing fat from WM, protein content in SM (4.29%, w/w)

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is relatively increased from that of WM (3.04%, w/w). Milk proteins such as casein and whey protein

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isolates are good encapsulation materials which stabilize materials by the free radical scavenging

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function of sulfhydryl and nonsulfhydryl amino acids.33 Thus, SM containing greater amounts of

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these proteins showed more stability than WM and the significant difference in k values between

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milk solutions and water may be due to the lack of proteins in the latter. Temperature is a critical

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factor in fucoxanthin degradation with several studies reporting fucoxanthin degradation with

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temperature increases.5, 9 Even though the temperature used in those experiments was generally room

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temperature, the trend of increasing fucoxanthin degradation with increasing temperature is an

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undoubted fact. This degradation effect was also demonstrated below room temperature as shown in

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Table 2. Regardless of the food matrices, degradation rate constant of fucoxanthin always showed 13



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increased tendency with temperature increases. Several isomers (13-cis, 13’-cis and 9-cis

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fucoxanthin) are reported (but not analyzed) as the degradation product of fucoxanthin in solution by

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thermal treatment.9

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Effect of Pasteurization Method on Fucoxanthin Stability. Pasteurization is an essential step in

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milk production and distribution that protects humans from infection of pathogenic bacteria.34 Until

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now, various pasteurization methods were developed and applied in the milk industry. The heat

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treatment method used for pasteurization can be categorized into three typical types based on the

304

temperatures and treatment times used: Low Temperature Long Time (LTLT), 65 ℃/30 min; Hot

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Temperature Short Time (HTST), 75 ℃/15 sec; Ultra-High Temperature (UHT), 135 ℃/2 sec. Table

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3 shows fucoxanthin recovery values over 91% after its treatment by these pasteurization steps,

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indicating that most fucoxanthin was retained during all tested pasteurization processes. In this

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experiment, the three factors of temperature, time, and food matrix are variables impacting the

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fucoxanthin recovery value. However, we did not determine any factors influencing consistently to

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fucoxanthin recovery value among these three variables in Table 3. Yet, this result is meaningful

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because it indicates that fucoxanthin is stable during the industrial process of pasteurization.

312 313

Effect of Drying Method on Fucoxanthin Stability. Three kinds of drying processes were also

314

applied to WM and SM solutions and investigated for fucoxanthin recovery. The drying process in

315

the milk industry is used to produce dried milk powder utilized in various foods. Among the tested

316

methods, the freeze-drying method demonstrated the highest recovery value and the hot air-drying

317

method showed the lowest recovery value (Fig. 3). Among WM and SM, the retained fucoxanthin

318

amounts were always high in SM. This result was similar with that of the stability test discussed

319

above. Thus, it could be assumed that the contents of fat and protein in both food systems influenced 14


Page 15 of 29


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fucoxanthin stability in the drying process. In both food systems, fucoxanthin recovery depended on

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temperature and treatment times during the drying processes. In terms of treatment times, hot air-

322

drying and freeze-drying methods required quite a long time (48 hr and 72 hr, respectively) and

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spray-drying required a relatively shorter time (below 10 second). In terms of temperature, there

324

were significant differences between each method (45 ℃ for hot air-drying, 108~180 ℃ for spray-

325

drying, -120 ℃ for freeze-drying method). Even though we could not determine either variable as

326

the more statistically significant factor, the mixed effect of these two factors in fucoxanthin recovery

327

was significant in both food systems.

328

In conclusion, fucoxanthin, a highly valuable marine carotenoid, was used to fortify two kinds of

329

milk solutions and its analytical method was developed and validated through LOD, LOQ, precision,

330

accuracy, system suitability and robustness tests. In addition, fucoxanthin stability was investigated

331

during storage, pasteurization, and drying processes in order to check the feasibility of fucoxanthin

332

application in milk products. As a result, the content of fats and milk proteins and temperature were

333

suggested as critical factors for fucoxanthin stability in these processes and it was proved that

334

fucoxanthin can be a suitable functional material for milk fortification. Thus, biological functions of

335

fucoxanthin such as anti-obesity activity can be expected from milk products fortified with

336

fucoxanthin. At present, fucoxanthin is not as popular as lutein and astaxanthin due to its short

337

supply in the global market. Nevertheless, this study suggests the plausibility of fucoxanthin

338

application to the food industry.

339 340

ACKNOWLEDGEMENT

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This research was supported by a grant from Marine Biotechnology Program (2MP0360) funded by

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Ministry of Oceans and Fisheries, Korea and an intramural grant (2Z04690) from KIST Gangneung 15



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Page 16 of 29

Institute of Natural Products

344 345

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Supporting Information. System suitability and robustness data of the developed fucoxanthin

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analysis method by HPLC-DAD. This material is available free of charge via the Internet at

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http://pubs.acs.org

349 350

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Figure captions

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Figure 1. The HPLC chromatogram (A) and comparison of fucoxanthin recovery values before and

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after n-hexane-partition (B) from fucoxanthin-fortified solutions. Initial fucoxanthin concentration of

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8 µg/mL was assumed as 100%.

＊P

Development, Quantification, Method Validation, and Stability Study of

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